Lessons learned in translating pain knowledge into practice

Using 3 molecular targets, the article explores the lack of translation of pain knowledge into the patient treatment and provides strategies to overcome these challenges.


Introduction
Molecular pain research is a relatively young field of a little more than 2 decades.Its beginning can be dated around 1995 with the discovery of P2X3 and Nav1.8 as genes enriched in dorsal root ganglion sensory neurons, partly expressing nociceptorassociated markers. 17,52Two further milestones to be mentioned are the discovery of the vanilloid receptors for noxious heat stimuli in 1997 16 and the piezo receptors for mechanical stimulation in 2010 18 -both receiving the Nobel Prize in Medicine in 2021.The Key Points 1. Several drugs targeting important pathways in pain transmission have not made it into the clinic.2. Reasons were the lack of efficacy in the disease entities studied or unexpected severe side effects.3. A package of measures will be necessary in the future, including the use of human material and more comprehensive preclinical data in several models using a battery of tests.4. Clinicians can help in the process by encouraging patients to participate in trials and reporting of side effects to the necessary authorities to raise awareness for postmarketing surveillance.5.While completely new drugs will need more time to come into the clinic, the pain field might benefit in the future from drugs discovered in other medical specialties.
2 decades have seen an explosive growth of investigations into the molecular architecture of primary afferents, the first neuronal link in the pathway that culminates in pain perception in the central nervous system (CNS).In parallel, biopsychosocial aspects of chronic pain have gained a scientific basis.These developments have advanced our understanding of the pain experience and identified new targets for the development of treatments for pain.Chronic pain arises from many different pathological conditions.These include primary chronic pain diseases like nonspecific back pain and fibromyalgia or secondary chronic pain as a sequela of cancer, an injury or damage to the somatosensory systems resulting in neuropathic pain.Diagnosis and treatment have remained a challenge.Opioids and nonopioids such as nonsteroidal anti-inflammatory drugs and paracetamol as well as selected antineuropathic drugs dominate daily routine in the clinic.However, they often present with limited effectiveness and considerable side effects, such as increased risk of falls and addiction for opioids and gastrointestinal bleeding and increased cardiovascular events for nonopioids.
There has been an intense effort by basic science researchers, clinicians, and pharmaceutical industry to understand pain at the molecular level and to identify new targets for drug development.This effort has faced multiple challenges.Beside the clinical trial design, that is not only an expensive but also a time-consuming process, one must consider different issues including-amongst others-careful section of patients, (active) placebos as controls, proper randomization, sufficient duration if patients with chronic pain are treated, and enough power to detect and effect and objective biomarkers when they become available. 8In addition, in the absence of validated biomarkers, measuring pain in humans is far from objective, too.In fact, the widely used pain ratings are strongly affected by social, cognitive, and emotional factors, such as suffering, social desirability, context, and expectations.
Another important hurdle in pain research is that although laboratory animals are sentient, they cannot directly communicate their pain "perception" in a language to be understood easily by humans.So, basic science researchers must refer to "nociceptive behavior" or "pain-related behavior," but this can also be highly affected by other variables, apparent or not well understood, which are not always taken into consideration.In addition, most of the models employed do not mirror the major clinical diseases.Pain is normally caused by impulses generated in the periphery, reaching a conscious brain.Although any treatment or drug may reduce paw withdrawal in a rodent animal model, this reflex response may not reflect a change in pain perception.This latter mechanism is hard to evaluate, although new and more sophisticated assays of affective components of pain are now expected by the research community and funding agencies.Finally, mice and men are different in the pain processing system, so extrapolation of preclinical data always carries risks, and the use of larger animal models and human tissues in these preclinical studies will hopefully bridge this knowledge gap.
Despite these limitations, some drugs have made it into the clinic including several anticonvulsants like gabapentin and pregabalin for neuropathic pain, albeit with limited efficiency, and antagonists or antibodies against calcitonin gene-related peptides (CGRP) for migraine. 29To date, 2 different classes targeting CGRP have been developed for migraine therapy: There are small molecule receptor antagonists of the CGRP receptor and blocking monoclonal antibodies.Their discovery started with CGRP measured in the blood of patients with migraine-so not entirely a bench to bedside.However, animal studies have confirmed the linkage of CGRP and migraine.Although the CGRP receptor is widely expressed, not only in the vascular tissues but also in the peripheral and central nervous system, no severe adverse effects, except elevation in transaminases in some subjects, have been reported to date.Therefore, CGRP receptor antagonists are a very effective and promising drug especially in migraine therapy. 21Other promising targets have not reached the clinic.Here, we will explore 3 drug targets and the lessons that they teach us.Out of the large group of molecules, we focused on 3 prominent ones from the group of growth factors, ion channels, and neuropeptides as targets-because they are highly relevant in the clinic.
2. Growth factors: nerve growth factor for the treatment of osteoarthritis and back pain 2.1.Nerve growth factor function and involvement in disease Nerve growth factor was discovered in the 1950s as a tumor tissue produced soluble factor influencing the somatosensory system. 13Nerve growth factor belongs to the group of neurotrophins containing 3 subunits, called a, b, and g. 58 During the embryonic development of the peripheral nervous system, nerve growth factor (NGF) directs differentiation of sensory and sympathetic neurons from the neuronal crest.In adulthood, NGF maintains skeletal muscle function and immune homeostasis for splenocytes.Nerve growth factor binds to 2 separate cell surface receptors: p75 and tropomyosin receptor kinase A (TrkA).Tropomyosin receptor kinase A has a high affinity for NGF, whereas p75 might function more as a coreceptor.When NGF binds to TrkA, the receptor dimerises and is phosphorylated and therefore results in the activation of the intracellular protein kinase.This results not only in an induced gene transcription of substance P (SP) and calcitonin gene-related peptide but also in an increased translocation of transient receptor potential vanilloid 1 (TRPV1) channel proteins in the cell surface membrane.Because of a positive feedback loop in the NGF signaling, this results in an increased NGF-induced hyperalgesia. 40Substance P and CGRP elicit vasodilation and chemotaxis, causing subsequent neurogenic inflammation.Second, binding of NGF to TrkA sensitized nociceptors for the activation of acid-sensing ion channels or transient receptor potential vanilloid TRPV1.

Clinical studies and challenges
After the initial discovery of NGF and its proalgesic properties in preclinical models, 57 the human equivalent was cloned, and selective NGF monoclonal antibodies were generated. 1They interfere with the functioning of TrkA-positive nociceptors and reduce their sensitivity to noxious stimuli. 37The first clinical osteoarthritis trial with tanezumab was conducted in 2010. 41Initial phase II and phase III clinical trials with NGF inhibitors were very promising and demonstrated efficacy not only in reducing joint pain but also in improving function. 49However, reports about a dosedependent rapidly progressive osteoarthritis (RPOA) soon emerged.Rapidly progressive osteoarthritis is characterized by pain, rapid joint space narrowing, and severe atrophic bone changes with collapse of the subchondral area within one year ultimately requiring joint replacement.Observation of this side effect led to a clinical hold by the FDA between 2010 and 2015. 50pparently, higher doses or a simultaneous application of nonsteroidal anti-inflammatory drugs contributed to this side effect. 58espite numerous efforts in subsequent analyses, it was not possible to predict by clinical, radiologic, or molecular biomarkers, which patients were at risk.Although there is no benefit for pain therapy in humans to be reported up to date, anti-NGF is approved by the European Medicines Agency for the treatment of painful arthritis in dogs (Librela|EuropeanMedicinesAgency(europa.eu)).So far, there are no reports about RPOA in this group of animals.Therefore, although science did not have any benefit for humans, this example shows how at least animal health can benefit from pain research. 27nfortunately, analgesic effectiveness and RPOA are both dose dependent.Possible explanations for RPOA suggest abnormal joint loading either due to the sensory loss to feel pain or due to analgesic effects to reduce joint pain. 58Good analgesia but joint destruction could be back translated to the preclinical models: anti-NGF treatment alters the microenvironment including innervation and blood flow within the joint leading to accelerated joint degeneration because NGF is expressed in endothelial cells in subchondral bone layers in close proximity of TrkA-positive sensory fibers.Indeed, it is known that nociceptors regulate blood flow, and mice that lack TrkA have reduced bone formation.

Summary and conclusions
In summary, NGF antibodies are the classical molecular pathway that showed good analgesic effectiveness, but the unexpected side effects-albeit not common but severe-finally prohibited its use.There are several explanations focusing on the development of RPOA.Beside the molecular mechanism, trial patients were also considered to overdo because of less pain sensation.Because reducing the dose led to a reduction in RPOA, it is to be assumed that unsuspected pathways led to these unwanted side effects.Reducing the dose of anti-NGF significantly reduced the incidence of RPOA, but it also showed significantly less effectiveness in pain therapy.In the future, this can only be avoided if systematic screenings are employed in preclinical models to detect surprising pathways.Nevertheless, studies are still ongoing for the treatment of other painful diseases in which RPOA is important, such as eg, schwannomatosis (Clinical Trials No NCT04163419) or bone cancer pain (eg, Clinical Trials No NCT02609828).
3. Ion channels: NaV1.7 blocker for the treatment of neuropathic pain

Nav1.7 function and involvement in disease
Voltage-gated sodium (Nav) channel Nav 1.7, which is abundant in peripheral sensory and sympathetic neurons, plays a critical role in action potential formation and has been genetically and functionally validated as causing human pain disorders. 9,25Fully penetrant gain-of-function mutations in Nav1.7 cause the human painful disorders like inherited erythromelalgia (IEM) and paroxysmal episodic pain disorder (PEPD).Less penetrant mutations have been identified in idiopathic adult-onset small fiber neuropathy and in patients with diabetic painful neuropathy. 9y contrast, loss-of-function mutations in Nav1.7 cause Nav1.7associated congenital indifference to pain (CIP) in which patients have a profound insensitivity to pain without any other apparent neurological abnormalities except anosmia. 9,25Nav1.7 has also been implicated in acquired pain after injury, inflammation, and diabetes. 24This has made this channel a prime target for the development of novel therapies for the treatment of pain.

Clinical studies and challenges of Nav1.7 blockers
There have been numerous clinical studies involving inhibitors of the Nav1.7 channel. 3Perhaps, the most extensively studied of these compounds are BIIB074 (also called vixotrigine) from Convergence/Biogen and PF-05089771 from Pfizer.
Vixotrigine is a state-dependent and use-dependent prolinamide blocker of Nav1.7, and thus, it is predicted to preferentially inhibit high firing neurons.However, it has now been shown not to be Nav1.7-selective. 22,35Vixotrigine received orphan drug status in 2013 and has been studied in 3 phase II clinical trials.A randomized, double-blind, placebo-controlled, crossover study that investigated the efficacy of vixotrigine in the treatment of neuropathic pain from lumbosacral radiculopathy in 82 subject 19 met the primary end point and significantly reduced daily neuropathic pain compared with baseline (P 5 0.0265).In another trial (2015), 6 Biogen also stated that the drug was more effective in individuals who were not on concomitant pain medications at the time of the study-they reported a more significant reduction in their pain compared with baseline. 33However, another 502person, 14-week study on patients with lumbosacral radiculopathy 56 did not meet its primary or secondary end points.The third phase II trial 5 was a multicenter, randomized, double-blind, placebo-controlled withdrawal trial for trigeminal neuralgia. 59The primary end point was treatment failure (defined as a 50% increase in either frequency of paroxysms or the intensity of pain during paroxysms).Although participants receiving vixotrigine experienced less treatment failure than placebo, 33.3% vs 64.3%, respectively, the result did not reach statistical significance.However, the secondary end points were met: vixotrigine reduced the number and severity of daily painful paroxysms, and the mean daily pain score compared with placebo. 59Biogen is no longer pursuing lumbosacral radiculopathy pain as an indication for vixotrigine; however will it continue to investigate it in trigeminal neuralgia 5 and has completed a phase II trial to treat small fiber neuropathy, but no results have been posted to date. 26he first-in-class arylsulfonamide PF-05089771 compound from Pfizer is a potent, selective, and peripherally restricted Nav1.7 antagonist. 2 A small proof-of-principle, double-blind, crossover, clinical study of PF-05089771 was conducted on 5 individuals with IEM.In preclinical studies using patient-specific sensory neurons differentiated from induced pluripotent stem cells (iPSC-SN), PF-05089771 reduced excitability of neurons from 3 of the 4 participants, suggesting potential efficacy in treating these patients. 14In the clinical study, participants received either a single oral dose of 1600 mg of PF-05089771 or placebo over 2 study periods, and their pain was triggered using a predetermined individualized thermal stimulus, which is characteristic of IEM. 14 The primary end point (pain relief during the 0-4 hours period after dosing) was not met-presumably because the necessary time of maximum concentration was not reached, unlike data from healthy volunteers in phase I studies.However, a statistically significant difference was observed at 8 to 9 h after dosing (P 5 0.03).In an interesting link to the preclinical study, PF-05089771 only reduced heat-evoked pain in the 3 participants whose iPSC-SN displayed reduced warmth-evoked hyperexcitability when exposed to the drug. 14F-05089771 was also assessed in a randomized, double-blind study in 135 individuals with diabetic peripheral neuropathy, 45 which evaluated the efficacy of the drug on its own and with pregabalin (a common treatment for peripheral neuropathy) to reduce pain from baseline in the PF-05089771 arm of the study after 4 weeks.A trend in pain reduction was reported, but this change was not statistically significant compared with placebo.The reduction in pain score from pregabalin was larger than that of PF-05089771 and statistically significant compared with placebo; the study did not continue to the second phase to evaluate PF-05089771 as an add-on to pregabalin.Another phase II trial with a 235-participant, randomized, double-blind, double-dummy, parallel group, placebo-controlled study assessed the efficacy of increasing doses of PF-05089771 in postoperative dental pain.43 The compound did not show superiority over ibuprofen.
The mismatch between preclinical and clinical data emphasized the need for a detailed knowledge of the plasma protein-bound and free-drug fractions, the ability to cross the blood-brain barrier and blood-nerve barrier, and the extent of target occupancy, which are crucial to understanding and predicting the efficacy of new drugs. 47rylsulfonamide Nav1.7 blockers, like PF-05089771, have limited CNS penetration. 45Because Nav1.7 is present at the central terminal of dorsal root ganglion neurons in the dorsal horn behind the blood-spinal cord barrier, 12 the block of Nav1.7 in the dorsal horn might be necessary to effectively inhibit pain signaling.There remains some uncertainty around the exact level of Nav1.7 blockade necessary to achieve analgesia.Using dynamic clamp electrophysiology and iPSC-SN, a recent study suggested that approximately 50% blockade of Nav1.7 currents can reverse neuronal hyperexcitability to baseline levels. 4A similar conclusion has also been reached using empirical data from rodents and nonhuman primates to model desirable pharmacocinetic/pharmacodynamic in humans for an effective target engagement. 7Although reduced excitability of neurons in culture might be a biomarker for an effective drug candidate, care must be exercised in equating it with reducing pain in patients.However, several animal studies suggested that channel occupancy by this class of Nav1.7 blockers required for efficacy might be higher than 50% and suggested the compound needed to achieve a plasma concentration of up to 50 times the IC 50 to achieve significant pain reduction. 31,55At these concentrations, the compounds could lose their selectivity and block other neuronal sodium channels.This challenge is likely related to a high percentage of plasma protein binding (.99%), high unbound clearance, and in vitro hepatotoxicity. 32,46

Summary and conclusions
The clinical trials to date have not yet revealed the Nav1.7-blockingmagic bullet for pain and have been hampered by mixed results.Although Vitroxigene is not Nav1.7-selective, it is well tolerated and could be useful if proven effective in the larger clinical trials.The new study by Kraus et al. 39 using a new arylsulfonamide Nav1.7 blocker with higher free-drug fraction points to improved pharmacokinetic properties of this class of molecules and is more effective than PF-05089771 in blocking conduction in sensory C-fibers and response to noxious heat in a nonhuman primate model, indicating that newer compounds in this class might overcome some of the challenges that affected the performance of PF-05089771 in clinical trials.This also suggests that the inhibition of Nav1.7 in the peripheral compartment might still be effective for pain treatment if sufficient free drug is present.There are also ongoing attempts to engineer and optimize naturally occurring Nav channel-targeting peptides, venoms 15,36 and toxins, like JNJ63955918 (which is a Protoxin-II derivative) or ceratoxin-1, 30,53 or subtype-specific antibodies. 11,44With these investigations underway, the development of Nav1.7-targeting drugs still maintains the promise of yielding new, safe, effective, and nonaddictive treatments for pain.

Tachykinin receptors and their involvement in disease
The tachykinin family of peptides (eg, substance P) interact through 3 neurokinin receptors (NKRs) encoded by 3 Tacr genes.
They are involved in several physiological and pathophysiological processes. 48,54Neurokinin 1 receptor (NK1R) (SP receptor) was cloned from a rat brain cDNA library by functional expression in Xenopus oocytes and cross-hybridization to bovine NK2R.It is generally suggested that these peptides are only released due to strong cell activation and that receptor antagonists can only act in a sensitized or pathological system. 54Therefore, tachykinin receptors have been of huge interest in the treatment of various disease conditions including chronic pain.
Substance P was first described as eliciting atropine resistant smooth muscle contraction and hypotension of the blood pressure.Substance P is widely distributed and can be found not only in the central and peripheral nervous system but also in nonneuronal cells, eg, mast cells and body fluids. 48Painful stimuli release SP from presynaptic terminals of nociceptors in the dorsal horn.Similarly, SP is released in the periphery by means of antidromic stimulation of direct activation, eg, by TRPV1 stimulation.Substance P binds neurokinin 1 receptor on postsynaptic neurons transmitting nociceptive signals to the brain.Troves of preclinical data with NK1R antagonists supported a key role for SP in chronic pain.Substance P does not activate an action potential in nociceptors per se but rather sensitizes nociceptors to other chemical, thermal, or mechanical stimuli inducing a left shift of the activation curve.

Clinical studies and challenges
Neuropeptides: tachykinin receptor 1 antagonists can be divided into 2 groups: peptide and nonpeptide antagonists.Due to negative side effects such as neurotoxicity, peptide antagonists are not employed in clinical practice. 48Up to date, 5 nonpeptide antagonists are on the market to treat nausea and vomiting in patients undergoing chemotherapy, 42 but none of them is approved for pain therapy.Clinical trials of highly selective NK1R antagonists were simply unsuccessful. 23,54Apparently, it is rather CGRP that mediates pain sensation while SP seems to be secondary. 54everal reasons can explain the ineffectiveness of NK1 receptor antagonists: species-specific differences in affinities of human vs preclinical animal models.Because most of the preclinical data came from rodents, lower affinity to the human NK1R is one of the reasons of failure.Because of the species differences, higher doses have been used, which could have led to other mechanisms of action including blockade of calcium influx.In addition, when NK1R antagonists were used in preclinical studies in the 1990s, disease mechanisms and molecular interactions were not comprehensively understood and the predictive value of animal pain behavior overestimated. 34,54For example, data are just beginning to emerge on molecular interaction of antagonist and receptor (allosteric/ orthosteric) and then inhibition vs activation in the presence of the agonists and interactions between NKR and G protein-coupled receptor (GPCR, heterodimers).Furthermore, it seems that there is an inherent redundancy of the tachykinin system, allowing for escape mechanisms. 54Recent evidence points toward an important role of the appropriate subcellular domain, eg, endosomal signaling. 38,43Because many GPCRtargeted drugs are not cell permeable, antagonists would block GPCR function at the cell surface but not within endosomes.

Summary and conclusions
Although substance P can sensitize nociceptors in vitro and in vivo, genetic ablation preclinical studies suggested that it might not play a major role in multiple pain modalities.These data should have raised some concern during the drug development process. 20Furthermore, our inadequate understanding of the localization of the NKR1 in subcellular organelles and how to target them could improve chances of future NK1R antagonists to succeed in clinical trials.Furthermore, unexpected side effects have been reported with current NK1R antagonists because most of them are metabolized through CYP3A4 and thus contribute to potential drug-drug interactions. 51So, NK1R antagonists might be better used to treat nausea and vomiting associated with a variety of conditions.A completely new field is the use of NK1R in the treatment of pruritus-based on observations in animal models-so new trials are on the way (Clinical Trials NCT03836001).

Discussion: lessons learned
When targeting a well-established pathophysiological mechanism fails to lead to new treatment, several lessons can be learnt.Failures to translate preclinical data can be caused by the lack of sufficient efficacy and severe side effects (Fig. 1).Here, we provided evidence for the former exemplified by targeting NK1R and NaV1.7 or severe side effects as seen for NGF antibodies.Other examples for the former also include TRPV1 and transient receptor potential ankyrin 1 (TRPA1) antagonists.Fatty acid amide hydrolase blockers like 3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide to prevent the degradation of endocannabinoids failed due to unexpected death in phase I clinical trials probably due to off target effects on the inhibition of other lipases. 28o enhance the success of translation, future studies should always include human tissue or human nociceptors from iPSCs to assess species-specific effects on the target.Patients should be phenotyped in more detail to make sure that only those who are treated with a drug that is specific to the target in this pathway-aiming toward an individual pathophysiology possibly also across different disease entities.Last but not least, measuring nociception as well as pain will be a challenge for the future in the treatment of chronic pain in its biopsychosocial context.Funding agencies should keep these aspects in mind when evaluating grants and their possible translation in the future.
Importantly, we need more specific biomarkers allowing to identify patients who could best benefit from a specific treatment.Postmarket monitoring by the appropriate institutions and examination of class effects are necessary steps in this direction.Patients and clinicians can support this process by reporting unexpected symptoms or diseases to these surveillance agencies.

Disclosures
H.L. Rittner recieved financial compensation from Algiax Pharmaceuticals GmbH, Gr ünenthal GmbH and Orion for consulting services.

Figure 1 .
Figure 1.Summary of reasons for failure for selected pathways and possible future direction of pain research to foster translation of pain knowledge into practice.Failure of analgesics in clinical trials can be attributed to 2 categories: lack of effect and unwanted side effects.Examples are given in the right column and explained in the main text.Many suggestions for the future have been made based on these observations-mainly to address the lack of effect.CCI, chronic constriction injury; CIPN, chemotherapy-induced neuropathy; CNS, central nervous system; DH, dorsal horn; DPN, diabetic polyneuropathy; DRG, dorsal root ganglion; FAAH, fatty acid amide hydrolase; IPSC, induced pluripotent stem cells; NaV1.7, voltage-gated sodium channel; NGF, nerve growth factor; RPOA, rapid progressing osteoarthritis; SNI, spared nerve injury; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid 1.